OC 38.2 - Neutrophils and platelets collaborate to induce an increase NET formation during JAK2V617F positive myeloproliferative neoplasms

Background: Thrombosis is a frequent complication during JAK2V617F myeloproliferative neoplasms (MPN) and neutrophils, especially through emission of neutrophil extracellular traps (NETs) were shown to promote thrombosis in MPN mouse models.

Aims: To assess whether JAK2V617F neutrophils alone are prone to NET formation, or whether they need to be activated to promote thrombosis.

Methods: We used two MPN mouse models with JAK2V617F expression in neutrophils: PF4-iCre;JAK2V617/WT mice with JAK2V617F expression in all blood cells compartments and MRP8-iCre;JAK2V617/WT mice with JAK2V617F expression only in neutrophils and monocytes. Thrombosis was analyzed in the lungs. Ex vivo NETosis was studied by immunofluorescence using histone 3 staining. In vivo NETosis was studied measuring plasmatic DNA and citrullinated histone 3 levels.

Results: We observed increased ex vivo NETosis in both models, but in vivo NETosis was only increased in PF4-iCre;JAK2V617/WT mice. Besides, only PF4-iCre;JAK2V617/WT mice had increased thrombosis formation, and NET inhibition reduced it. We hypothesized that increased inflammation or increased platelet activation could explain the differences in NET formation and thrombosis between PF4-iCre;JAK2V617/WT and MRP8-iCre;JAK2V617/WT mice. We could not find any evidence for the role of inflammation as (1) plasmatic TNF-alpha concentration was similar, (2) addition of plasma from PF4-iCre;JAK2V617/WT mice on JAK2V617F neutrophils did not increase ex vivo NETosis, (3) TNF-alpha or LPS treatment of MRP8-iCre;JAK2V617/WT mice did not increase thrombosis. We then focused on platelets and observed increased ex vivo NETosis when JAK2V617F platelets were coincubated with JAK2V617F neutrophils. Finally, treatment of PF4-iCre;JAK2V617/WT mice with aspirin reduced in vivo NETosis and thrombosis formation.

Conclusion(s): We show here that the presence of JAK2V617F neutrophils together with JAK2V617F platelets is necessary to trigger increased NETosis and thrombosis in a mouse model of JAK2V617F MPN. Interestingly, platelet inhibition can inhibit NETosis and subsequent increased thrombosis formation.